Project 3: Attenuating RSV with Mutations in Its Methyltransferase PI: Jianrong Li, DVM, PhD Abstract: Human respiratory syncytial virus (RSV) is the leading causative agent of pediatric respiratory tract disease worldwide. Despite major efforts, there is no vaccine currently available to combat this virus. Live attenuated vaccines are the most promising vaccine candidates for RSV. However, it has been a challenge to identify an attenuated RSV strain that has an optimal balance between attenuation and immunogenicity. The objectives of this project are to rationally attenuate RSV by targeting the viral mRNA cap methyltransferase (MTase) and to develop MTase-defective viruses as novel live attenuated vaccine candidates for RSV. The RSV mRNA cap structure is methylated at the guanine-N-7 (G-N-7) and ribose 2?-O positions by a single conserved region VI (CR-VI) located in the large (L) polymerase protein. Amino acid residues essential for G-N-7 and/or 2?-O MTase activities will be characterized and will be introduced into an infectious cDNA clone of RSV. Recombinant RSVs defective in G-N-7 and/or 2?-O MTase activities will be recovered, and their attenuation in cell culture will be characterized. These recombinant viruses will be tested for their infectivity and ability to spread in primary, well differentiated, human airway epithelial (HAE) cultures. Next, their replication, pathogenesis, immunogenicity, and capacity to induce protection will be examined in cotton rats, the best available small animal model for RSV. By adding interferon-producing NS1 mutations (Project 1) and ?repaired G? mutations (Project 2) to the most promising MTase mutations in L to RSV, we expect that we will generate a panel of ideal RSV vaccine strains that grow well in Vero cells, are attenuated in vitro and in vivo, genetically stable, and highly immunogenic. These new, improved live RSV vaccine candidates can then be used for future trials in nonhuman primates.